Lecture 22: Molecular techniques DNA cloning and DNA libraries
DNA cloning: general strategy -> to prepare large quantities of identical DNA Vector + DNA fragment Recombinant DNA (any piece of DNA derived from different sources) Replication within host cells Isolation, sequencing, manipulation of purified DNA fragment
DNA cloning: Vectors Bacteria (Escherichia coli) Most common host cell Plasmids up to ~ 10 kb λ bacteriophages/cosmids id up to ~ 50 kb Bacterial artificial chromosomes (BACs) up to ~ 2Mb Yeast (Saccharomyces cerevisiae) Plasmids Yeast artificial chromosomes
DNA cloning: host cells: bacterial cultures agar plate Petri dish Soft solid support with nutrients Liquid culture Pick clone Grow bacteria individual E.coli colonies Clone: start from one single bacterium Purify plasmid DNA from bacteria
DNA cloning: Enzymes Cut and paste Restriction enzymes DNA ligases
cut DNA cloning cut VECTOR (plasmid) INSERT paste OPEN VECTOR NEW RECOMBINANT DNA
DNA cloning: Restriction enzymes Restriction site = palindomic GAATTC Single-stranded tail = overhang (can be 3 or 5 ) Blunt ends: ex. SmaI CCCGGG GGGCCC CCC GGG GGG CCC
DNA cloning: Restriction enzymes 5 overhang 5 overhang 5 overhang 3 overhang
DNA cloning: Restriction enzymes 3 overhang 3 overhang 5 overhang 5 overhang 5 overhang
DNA cloning: Restriction map All sites From a large list of enzymes and corresponding DNA sequences EcoRI BamHI XbaI SmaI NotI XbaI EcoRV ClaI SmaI BglII XbaI 145 159 180 221 Unique sites Restriction digestion BamHI EcoRV Restriction fragment
DNA cloning: Ligation ( ) ( )
DNA cloning: Ligation!Blunt ->less efficient!
DNA cloning: Engineered E.coli plasmid Stripped from unessential sequences ->1.2-3 kb
DNA cloning: Engineered E.coli plasmid Stripped from unessential sequences ->1.2-3 kb Origin of replication Antibiotic resistance (ampicillin, kanamycin) = gene! or multiple cloning site
DNA cloning: Introducing DNA in cells 1. Insertion in vector + ORI
DNA cloning: Introducing DNA in cells 2. Transformation and selection low efficiency of transformation!
DNA cloning: Introducing DNA in cells 3. Replication
DNA cloning: Introducing DNA in cells 4. Growth Always in the presence of antibiotics, or else the plasmid will be lost
DNA cloning: Introducing DNA in cells transformation selection agar plate (growth medium + antibiotic) pick single clone individual E.coli colonies containing plasmid grow in liquid medium isolate plasmid DNA keep transformed bacteria (e.g. frozen)
Preparation of a cdna library using reverse transcriptase I Poly A = characteristic of mrnas mrna AAAAAAA 5 3 hybridize polyt primer 5 AAAAAAA3 Poly T primer used for reverse 3 TTTTTTTT5 transcription starting from the tail reverse transcriptase of mrna 5 AAAAAAA3 3 TTTTTTTT5 DNA digestion of RNA with RNases H 5 3 TTTTTTTT5 synthesis of complementary strand using leftover etove RNA fragments tsasp primers es 5 AAAAAAA3 3 TTTTTTTT5 5 end of original mrna cdna often absent from cdna library
Preparation of a cdna library using reverse transcriptase II mrna DNA AAAAAAA 5 3 hybridize polyt primer 5 AAAAAAA3 3 TTTTTTTT5 reverse transcriptase 5 AAAAAAA3 3 TTTTTTTT5 5 end of original mrna can still be absent from cdna library remove RNA, and ligate?? synthetic oligonucleotide and anneal complementary primer synthesis of complementary strand using gcomplementary pe e tayprimer cdna TTTTTTTT5 AAAAAAA3 TTTTTTTT5
Supplementary slide: Preparation of full length cdnas using reverse transcriptase During extraction mrnas can be partially degraded. The use of polyt primers allows selection of mrna with a complete 3 end. However, in many cases the most 5 part may also be missing. To prepare a cdna library containing exclusively copies of full length mrnas, new protocols include a initial treatment of the mrna to selectively add a primer to the 5 of the mrna BEFORE reverse transcriptase. The strategy is based on the fact that the m7g cap is resistant to dephosphorylation by phosphatases. Phosphatase treatment will thus remove the 5 phosphate from all incomplete mrnas. The next step, ligation, requires the presence of a 5 phosphate (see ligation), and thus will work only on full length mrna still containing the cap (m7g) with its phosphate. The only cdnas that will include a copy of the primer are thus full length, which can then be selected by further synthesis with complementary primer. modified mrna mrna mrna 5 AAAAAAA3 m7 5 G AAAAAAA3 select cap-containing mrnas (resistant to dephosphorylation), ligate primer m7 G 5 AAAAAAA3 reverse transcriptase TTTTTTT 3 Synthesize double strand with complementary primer TTTTTTT 3
cdna library chromosome mrna 1 mrna 2 mrna 3 Cell extract: Mixture of all mrnas cdna (mixture) Reverse transcription Clone in plasmids -> transform in bacteria -> cdna library = contain coding regions!
genomic library chromosome Mixture of chromosome fragments Clone in bacteriophages/cosmids -> propagated by infecting bacteria or in BACs = bacterial artificial chromosomes -> up to 20 Mb! -> genomic library
Genomic/ cdna libraries Genomic library exon exon exon cdna library coding sequences exon promoter/regulatory sequence
Library screening Oligonucleotide probe: Short: chemical synthesis Long: PCR 40-65 o C 0.3-0.6M NaCl
Library screening same protocol can be used with a plasmid library agar plate lt individual E coli individual E.coli colonies
Expression of recombinant proteins Origin of replication cdna Antibiotic resistance (ampicillin, kanamycin) or multiple cloning site
Expression of recombinant proteins Is this enough to PROMOTER! drive expression of a gene? cdna or multiple promoter pp cloning site Promoter required for transcription of the gene
Production of recombinant protein in bacteria Inducible system using lacz promoter Lactose analogue
Production of recombinant protein in bacteria Coding region for gene of interest (cdna) mrna recombinant
Expression of recombinant protein in eukaryotic cells
Expression of recombinant protein in eukaryotic cells Neomycin resistance G418 = Neomycin analogue
Detection of epitope-tagged recombinant protein Epitope tag 5 ATG GAC GTG CCA GAC TAT GCT TCT TTT AAT 3 Met Asp Val Pro Asp Tyr Ala Ser Leu HA-tag Anti-HA antibody Tag sequence can be added d at the 5 or 3 end of the cdna
Detection of epitope-tagged recombinant protein Immunofluorescence of transiently transfected cells green red Adapter protein involved in intracellular vesicle transport Golgi marker Yellow = colocalization